Approximately 40,000 women die from breast cancer every year. The extent of lymph node metastasis is a major determinant for staging and prognosis of this disease. Clinical and experimental evidence suggests that migration of cancer cells into lymph nodes is also mediated by lymphangiogenesis, a process that generates new lymphatic vessels from pre-existing ones. Nevertheless, the molecular mechanisms of tumor-induced lymphangiogenesis are not well understood. Sphingosine-1-phosphate (S1P), a bioactive lipid mediator and a ligand for specific S1P receptors regulates critical processes of breast cancer progression, such as cell proliferation, migration and angiogenesis. Sphingosine kinase 1 (SphK1), the enzyme that generates S1P, is up-regulated in breast cancer and is associated with resistance to chemotherapy and correlates with a poor prognosis. We have recently shown that ATP-binding cassette transporters export S1P from breast cancer cells, and subsequently activate its receptors by inside-out signaling. Our preliminary studies suggest that S1P also plays an important role in tumor-induced lymphangiogenesis. We have found that S1P induces lymphangiogenesis via the S1PR1 receptor on lymphatic endothelial cells (LECs) and it stimulates these cells to release angiopoietin 2 (Ang2), a critical regulator of lymphangiogenesis, whose levels in breast cancer correlate with metastases to lymph nodes and poor prognosis. The goal of this proposal is to determine the role of SphK1/S1P/S1PR1 axis and its crosstalk with Ang2 pathway to form a feed-forward amplification loop and explore its possibility as a target for treatment. We will examine the hypothesis that S1P produced by SphK1 is a previously uncharacterized cancer-induced lymphangiogenic factor that is critical for breast cancer progression and metastasis. Pharmacological, molecular biological and extensive genetic approaches will be utilized to determine the intricate crosstalk between S1P, released from the primary tumor and/or LECs, and Ang2, released from LECs via the S1PR1 receptor to form a feed-forward amplification loop, and evaluate their important roles in breast cancer-induced lymphangiogenesis. We will further elucidate their impact in lymph node metastasis and reduced survival utilizing our validated syngeneic breast cancer metastasis model, and explore clinical evidence using patient samples to support our hypothesis. Collectively, these studies will lead to better understanding of the role of SphK1/S1P/S1P1 axis in lymphangiogenesis and breast cancer biology that could lay the foundation for the addition of a novel S1P-targeted modality of treatment to the armamentarium of anti-breast cancer agents. PUBLIC HEALTH RELEVANCE: Breast cancer kills patients because they spread (cause metastasis) starting from lymph nodes, which is one of three major determinants for staging and prognosis. This proposal will determine the role of sphingosine-1-phosphate (S1P), a bioactive lipid mediator, in the formation of new lymphatic vessels (lymphangiogenesis) associated with breast cancer metastasis. Based upon our hypothesis, we will explore the effects of pharmacological and molecular approaches that target S1P production and signaling in cells and animal models, which we expect to, provide us with preclinical data leading to novel targeted therapies against breast cancer metastasis.